Chemokines are small proteins involved in cell migration. The chemokine CXCL12
(SDF-1) signals via the receptor CXCR4. CXCR4 is overexpressed in more than 20 types
of cancer, and associated with metastasis and increased cancer cell growth and survival.
The diversity and complexity of CXCR4 signaling are subject to research, but CXCR4
signals through two major pathways: Gαi and β-arrestin 2. Regulatory mechanisms for β-
arrestin recruitment remain to be defined, but the recent interpretive framework describes
a two-stage recruitment mechanism allowing for two levels of interaction. The first step
would allow binding of the C-terminal end of the receptor to β-arrestin, forming a partial
interaction, while the second would allow subsequent binding of β-arrestin to the core of
the receptor, forming a complete interaction.
This project studies these different recruitment modalities using the constitutively
active mutants of CXCR4; N119S and R134A, using bioluminescence resonance energy
transfer (BRET) systems. We confirmed that CXCR4 N119S is a constitutively active
mutant (CAM) on both Gαi and the arrestin pathway. On the other hand, R134A is a
constitutively inactive mutant (CIM), thus lacking signaling by the G protein, but
spontaneously recruits β-arrestin.
We suggest that the differences in recruitment of β-arrestin observed in BRET
between these two mutants represent these different recruitment modalities. Thus, the
mutant N119S represents the complete interaction while the mutant R134A represents the
partial interaction. Similarly, we observe differences between these mutants in activation
of ERK by Immunoblot. Finally, the use of PTX, inhibiting the activation of Gαi,
demonstrates that the mutant R134A recruits β-arrestin through a different modality. We
suggest that this mutant allows the formation of a megaplex between β-arrestin and the
heterotrimeric G-protein.